Pharmaceutical composition for treating and/or preventing cancer

ABSTRACT

The present invention provides a novel pharmaceutical composition for treating and/or preventing a cancer comprising, as an active ingredient, a polynucleotide derived from various miRNAs associated with cancer, a combination drug of the pharmaceutical composition and another antitumor agent, and a method for treating or preventing a cancer in a subject having the cancer using the pharmaceutical composition or the combination drug. The present invention relates to a pharmaceutical composition for treating and/or preventing a cancer comprising, as an active ingredient, a polynucleotide comprising the nucleotide sequence as set forth in SEQ ID NO: 1 or 2 (wherein when at least a part of the polynucleotide is DNA, uracil in a region corresponding to the DNA in the nucleotide sequence is replaced with thymine).

TECHNICAL FIELD

The present invention relates to a pharmaceutical composition fortreating and/or preventing a cancer comprising, as an active ingredient,a polynucleotide derived from microRNA.

BACKGROUND ART

MicroRNA (miRNA) is an RNA of 16 to 28 nucleotides that is nottranslated into a protein, and it is currently known that 2590 miRNAsare present in human according to the miRBase release 21(http://www.mirbase.org/). In recent years, miRNAs have been receivingattention as molecules for suppressing in vivo expression of variousgenes. On the genome, a region of each of miRNA gene is present and istranscribed into a RNA precursor with hairpin loop by RNA polymerase II,and the RNA precursor is then cleaved by two types of dsRNA cleavingenzymes having RNase III cleavage activities that are called Drosha inthe nucleus and Dicer in the cytoplasm, thereby forming a mature miRNA.It is known that the mature miRNA is taken into the protein complexcalled RISC and interacts with mRNAs of a plurality of target geneshaving complementary sequences to suppress the expression of a gene (NonPatent Literature 1).

Certain types of miRNAs are suggested to be associated with humandiseases including cancer, and particularly in cancer, for examples,many miRNAs such as hsa-miR-4450, hsa-miR-6893-5p and hsa-miR-575 areknown to be markers specific to pancreatic cancer in blood (Non PatentLiterature 1 and Patent Literature 1).

Further, in addition to the miRNAs associated with the growth of cancercells, the presence of a miRNA which works in a direction of suppressingcancer cells is reported, suggesting a method for treating cancerutilizing the expression pattern of the miRNA. Specific examples of theknown method include a method for treating diseases such as cancer byadministering an activated serum comprising 153 miRNAs such ashsa-Let-7a and upregulating the miRNA (Patent Literature 2), a methodfor treating lung cancer using a body fluid comprising many miRNAs suchas hsa-Let-7a (Patent Literature 3), and a method for treating bloodcancer by administering antisense oligonucleotides of many miRNAs suchas miR-1321 comprised in circulating exosomes in the body (PatentLiterature 4).

CITATION LIST Patent Literature

-   Patent Literature 1: International Publication No. WO 2015/182781-   Patent Literature 2: International Publication No. WO 2011/029903-   Patent Literature 3: International Publication No. WO 2014/072468-   Patent Literature 4: International Publication No. WO 2014/071205

Non Patent Literature

-   Non Patent Literature 1: Kojima M PLoS One. 10(2) (2015) “MicroRNA    markers for the diagnosis of pancreatic and biliary-tract cancers.”

SUMMARY OF INVENTION Technical Problem

It is an object of the present invention to identify a miRNA exhibitingtherapeutic and/or preventive effects, in common, on various types ofcancers, among various cancer-relating miRNAs, and to provide a novelpharmaceutical composition for treating and/or preventing cancercomprising, as an active ingredient, a polynucleotide derived from themiRNA.

Solution to Problem

The present inventors conducted extensive studies to solve theaforementioned problem and have now found a novel polynucleotide, whichsuppresses the growth of cancer cells, among miRNAs with increased ordecreased expression in body fluids or tissues of cancer patients,thereby completing the present invention.

Specifically, the present invention encompasses the following (1) to(9).

(1) A pharmaceutical composition for treating and/or preventing a cancercomprising, as an active ingredient, a polynucleotide comprising thenucleotide sequence as set forth in SEQ ID NO: 1 or 2.

(2) The pharmaceutical composition according to the above (1), whereinthe polynucleotide is 8 to 60 nucleotides in length.

(3) The pharmaceutical composition according to the above (1) or (2),wherein the polynucleotide comprises a nucleotide sequence of thefollowing (a) or (b) on the 3′ terminal side of the nucleotide sequenceas set forth in SEQ ID NO: 1 or 2:

-   -   (a) the nucleotide sequence as set forth in any one of SEQ ID        NOs: 3 to 5, or    -   (b) a nucleotide sequence comprising a deletion, substitution,        insertion, and/or addition of 1 to 5 nucleotides in the        nucleotide sequence as set forth in any one of SEQ ID NOs: 3 to        5.        (4) The pharmaceutical composition according to any one of the        above (1) to (3), wherein the polynucleotide comprises the        nucleotide sequence as set forth in any one of SEQ ID NOs: 6 to        9.        (5) The pharmaceutical composition according to any one of the        above (1) to (4), wherein the polynucleotide is single stranded        or double stranded.        (6) The pharmaceutical composition according to any one of the        above (1) to (5), wherein the polynucleotide is RNA.        (7) The pharmaceutical composition according to any one of the        above (1) to (6), wherein the cancer is a solid cancer.        (8) The pharmaceutical composition according to the above (7),        wherein the cancer is selected from the group consisting of        breast cancer, kidney cancer, pancreatic cancer, colorectal        cancer, lung cancer, brain tumor, stomach cancer, cervical        cancer, ovarian cancer, prostate cancer, bladder cancer,        esophagus cancer, liver cancer, fibrosarcoma, mast cell tumor,        and melanoma.        (9) The pharmaceutical composition according to any one of the        above (1) to (8), wherein the polynucleotide is inserted into a        vector in an expressible manner in the form of DNA.        (10) The pharmaceutical composition according to any one of the        above (1) to (9), wherein the polynucleotide is encapsulated        into a carrier selected from the group consisting of        non-cationic polymer carriers, liposome carriers, dendritic        carriers, nano-material carriers, microparticle carriers,        biostructural carriers, micelle carriers, polymer        microparticles, and magnetic microparticles; or the        polynucleotide is bound to the carrier.        (11) A combination drug for treating and/or preventing a cancer        comprising, as active ingredients, the pharmaceutical        composition according to any one of the above (1) to (10), and        an antitumor agent.        (12) A method for treating or preventing a cancer in a subject        who suffers or has suffered from the cancer, comprising        administering the pharmaceutical composition according to any        one of the above (1) to (10), or the combination drug according        to the above (11), to the subject.

The present description includes the contents as disclosed in JapanesePatent Application No. 2015-240951 from which the present applicationclaims priority.

Advantageous Effects of Invention

The pharmaceutical composition for treating and/or preventing cancer ofthe present invention effectively suppresses the growth of cancer cells,and therefore it is useful for treating or preventing cancer.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 This figure shows the ratios of viable cell counts (cellviability (%)) of the pancreatic cancer cell line Panc-1 after theintroduction of a synthetic RNA having the same nucleotide sequence ashsa-miR-4450 as set forth in SEQ ID NO: 6 or a synthetic RNA having thesame nucleotide sequence as hsa-miR-8053 as set forth in SEQ ID NO: 7into the cancer cells, relative to the viable cell count (100%) afterthe introduction of a synthetic RNA being a negative control oligo intothe cancer cells.

FIG. 2 This figure shows the ratios of viable cell counts (cellviability (%)) of the breast cancer cell line MCF-7 after theintroduction of a synthetic RNA having the same nucleotide sequence ashsa-miR-4450 as set forth in SEQ ID NO: 6 or a synthetic RNA having thesame nucleotide sequence as hsa-miR-8053 as set forth in SEQ ID NO: 7into the cancer cells, relative to the viable cell count (100%) afterthe introduction of a synthetic RNA being a negative control oligo intothe cancer cells.

FIG. 3 This figure shows the ratios of viable cell counts (cellviability (%)) of the lung cancer cell line A549 after the introductionof a synthetic RNA having the same nucleotide sequence as hsa-miR-4450as set forth in SEQ ID NO: 6 or a synthetic RNA having the samenucleotide sequence as hsa-miR-8053 as set forth in SEQ ID NO: 7 intothe cancer cells, relative to the viable cell count (100%) after theintroduction of a synthetic RNA being a negative control oligo into thecancer cells.

FIG. 4 This figure shows the ratios of viable cell counts (cellviability (%)) of the stomach cancer cell line NC1-N87 after theintroduction of a synthetic RNA having the same nucleotide sequence ashsa-miR-4450 as set forth in SEQ ID NO: 6 or a synthetic RNA having thesame nucleotide sequence as hsa-miR-8053 as set forth in SEQ ID NO: 7into the cancer cells, relative to the viable cell count (100%) afterthe introduction of a synthetic RNA being a negative control oligo intothe cancer cells.

FIG. 5 This figure shows the ratios of viable cell counts (cellviability (%)) of the liver cancer cell line HepG2 after theintroduction of a synthetic RNA having the same nucleotide sequence ashsa-miR-4450 as set forth in SEQ ID NO: 6 or a synthetic RNA having thesame nucleotide sequence as hsa-miR-8053 as set forth in SEQ ID NO: 7into the cancer cells, relative to the viable cell count (100%) afterthe introduction of a synthetic RNA being a negative control oligo intothe cancer cells.

FIG. 6 This figure shows the ratios of viable cell counts (cellviability (%)) of the colorectal cancer cell line HCT116 after theintroduction of a synthetic RNA having the same nucleotide sequence ashsa-miR-4450 as set forth in SEQ ID NO: 6 or a synthetic RNA having thesame nucleotide sequence as hsa-miR-8053 as set forth in SEQ ID NO: 7into the cancer cells, relative to the viable cell count (100%) afterthe introduction of a synthetic RNA being a negative control oligo intothe cancer cells.

FIG. 7 This figure shows the ratios of viable cell counts of thecolorectal cancer cell line HCT116 after the introduction of a syntheticRNA having the same nucleotide sequence as hsa-miR-4450 as set forth inSEQ ID NO: 6 (present invention), a synthetic RNA having the nucleotidesequence as set forth in SEQ ID NO: 8 (present invention), a syntheticRNA having the nucleotide sequence as set forth in SEQ ID NO: 9 (presentinvention), or a synthetic RNA having the same nucleotide sequence ashsa-miR-4454 as set forth in SEQ ID NO: 10 (comparative example) intothe cancer cells, relative to the viable cell count (100%) after theintroduction of a synthetic RNA being a negative control oligo into thecancer cells (the cell viabilities of 4%, 18%, 4%, and 100%,respectively).

FIG. 8 This figure shows the ratios of viable cell counts of thecolorectal cancer cell line HCT116 after the introduction of a syntheticRNA having the same nucleotide sequence as hsa-miR-4450 as set forth inSEQ ID NO: 6 (present invention), a synthetic RNA having the samenucleotide sequence as hsa-miR-8053 as set forth in SEQ ID NO: 7(present invention), a synthetic RNA having the same nucleotide sequenceas hsa-miR-575 as set forth in SEQ ID NO: 11 (comparative example), or asynthetic RNA having the same nucleotide sequence as hsa-miR-1321 as setforth in SEQ ID NO: 12 (comparative example) into the cancer cells,relative to the viable cell count (100%) after the introduction of asynthetic RNA being a negative control oligo into the cancer cells (thecell viabilities of 4%, 46%, 96%, and 93%, respectively).

FIG. 9 This figure shows the ratio of viable cell count (cell viability:93%) of the mammary epithelial cell line 184B5 (normal cells) after theintroduction of a synthetic RNA (3 nM) having the same nucleotidesequence as hsa-miR-4450 as set forth in SEQ ID NO: 6 into the cancercells, relative to the viable cell count (100%) after the introductionof a synthetic RNA being a negative control oligo into the normal cells.

FIG. 10 This figure shows (B) changes in tumor volume for 13 days and(A) the tumor volume ratio on day 13, after the administration of asynthetic RNA having the same nucleotide sequence as hsa-miR-4450 as setforth in SEQ ID NO: 6 or a synthetic RNA being a negative control oligoto tumor-bearing mice.

DESCRIPTION OF EMBODIMENTS

<Polynucleotide as Active Ingredient>

The pharmaceutical composition for treating and/or preventing a cancerof the present invention comprises, as an active ingredient, apolynucleotide comprising the nucleotide sequence of UGGGGAUU (SEQ IDNO: 1) or UGGCGAUU (SEQ ID NO: 2). Hereinafter, the polynucleotide as anactive ingredient in the present invention will be described.

The nucleotide sequence as set forth in SEQ ID NO: 1 is a nucleotidesequence identified as a partial sequence on the 5′ terminal side ofhsa-miR-4450 (miRBase Accession No. MIMAT0018971), which is a humanmiRNA. The nucleotide sequence as set forth in SEQ ID NO: 2 is anucleotide sequence identified as a partial sequence on the 5′ terminalside of hsa-miR-8053 (miRBase Accession No. MIMAT0030980), which is ahuman miRNA. Among these miRNAs, hsa-miR-4450 has been known as a partof miRNA serving as a specific marker for pancreatic cancer (Kojima M.,PLoS One, 10(2) (2015) “MicroRNA markers for the diagnosis of pancreaticand biliary-tract cancers.”). However, the present inventors have foundfor the first time that the miRNAs suppress the growth of pancreaticcancer cells and other cancer cells, and that the polynucleotide havingthe nucleotide sequence as set forth in SEQ ID NO: 1 or 2, which is apartial sequence of either of these miRNAs, plays an important role insuppressing the growth of cancer cells.

Thus, the polynucleotide of the present invention is not particularlylimited, as long as it comprises the nucleotide sequence as set forth inSEQ ID NO: 1 or 2. Specifically, the polynucleotide of the presentinvention may be the nucleotide sequence as set forth in SEQ ID NO: 1 or2 itself, or a polynucleotide comprised of the nucleotide sequence asset forth in SEQ ID NO: 1 or 2 and another nucleotide sequence added tothe 5′-terminal or 3′-terminal side thereof. The polynucleotide of thepresent invention is preferably a polynucleotide comprised of thenucleotide sequence as set forth in SEQ ID NO: 1 or 2 and anothernucleotide sequence added to the 3′-terminal side thereof. Thepolynucleotide of the present invention is preferably 8 to 60nucleotides in length, and more preferably 16 to 28 nucleotides inlength.

The nucleotide sequence to be added to the 3′-terminus of the nucleotidesequence as set forth in SEQ ID NO: 1 or 2 is preferably a nucleotidesequence comprising the following (a) or (b) as a partial sequence, morepreferably a nucleotide sequence comprising the following (a) or (b) onthe 5′-terminal side thereof, and further preferably a nucleotidesequence consisting of the following (a) or (b):

(a) the nucleotide sequence as set forth in any one of SEQ ID NOs: 3 to5, or

(b) a nucleotide sequence comprising a deletion, substitution,insertion, and/or addition of 1 to 5 nucleotides, preferably 1 to 4nucleotides, more preferably 1 to 3 nucleotides, further preferably 1 or2 nucleotides, and particularly preferably 1 nucleotide, in thenucleotide sequence as set forth in any one of SEQ ID NOs: 3 to 5.

In a preferred embodiment, the polynucleotide of the present inventionmay consist of the nucleotide sequence as set forth in SEQ ID NO: 1 or 2and the nucleotide sequence of the above (a) or (b) added to the3′-terminus or 5′-terminus thereof.

When at least a part of the polynucleotide of the present invention isDNA, uracil in a region corresponding to the DNA in the nucleotidesequence as set forth in SEQ ID NO: 1, 2, 3, 4 or 5 is replaced withthymine.

Preferred specific examples of the polynucleotide in which anothernucleotide sequence is added to the 3′-terminal side of the nucleotidesequence as set forth in SEQ ID NO: 1 or 2, include polynucleotidescomprising the nucleotide sequences as set forth in SEQ ID NOs: 6 to 9(wherein when at least a part of the polynucleotide is DNA, uracil in aregion corresponding to the DNA in the nucleotide sequences as set forthin SEQ ID NOs: 6 to 9 is replaced with thymine). Particularly preferredexamples of such polynucleotides include polynucleotides consisting ofthe nucleotide sequences as set forth in SEQ ID NOs: 6 to 9 (whereinwhen at least a part of the polynucleotide is DNA, uracil in a regioncorresponding to the DNA in the nucleotide sequences as set forth in SEQID NOs: 6 to 9 is replaced with thymine). Among these four types ofpolynucleotides, the polynucleotide comprising the nucleotide sequenceas set forth in SEQ ID NO: 6 or 7 is known as miRNA that has alreadybeen identified in humans. The names and miRBase Accession Nos.(registration numbers) of these miRNAs are as shown in Table 1.

TABLE 1 SEQ ID NO: Gene name miRBase Accession No. 6 hsa-miR-4450MIMAT0018971 7 hsa-miR-8053 MIMAT0030980 10 hsa-miR-4454 MIMAT0018976 11hsa-miR-575 MIMAT0003240 12 hsa-miR-1321 MIMAT0005952

hsa-miR-4450, which is a miRNA having the nucleotide sequence as setforth in SEQ ID NO: 6, is composed of the nucleotide sequence as setforth in SEQ ID NO: 1 as a nucleotide sequence from the 5′ terminus(position 1) to the 8th nucleotide, and the nucleotide sequence as setforth in SEQ ID NO: 3 as the 9th and the subsequent remainingnucleotides. This miRNA has been known as a specific marker forpancreatic cancer, as described above. However, it has not been reportedso far that compounds based on the sequence of a gene of this miRNA or atranscript thereof can suppress tumor cells.

hsa-miR-8053, which is a miRNA having the nucleotide sequence as setforth in SEQ ID NO: 7, is composed of the nucleotide sequence as setforth in SEQ ID NO: 2 as a nucleotide sequence from the 5′ terminus(position 1) to the 8th nucleotide, and the nucleotide sequence as setforth in SEQ ID NO: 4 as the 9th and the subsequent remainingnucleotides. This miRNA has been identified by the method described inWang H J et al., 2013, Shock., No. 39, pp. 480-487. In addition, theprecursor of hsa-miR-8053 is known as hsa-mir-8053 (miRBase AccessionNo. MI0025889), which has a hairpin-like structure. However, it has notbeen reported so far that compounds based on the sequence of a gene ofthis miRNA or a transcript thereof can suppress tumor cells.

A polynucleotide consisting of the nucleotide sequence as set forth inSEQ ID NO: 8 is an artificial polynucleotide in which the nucleotidesequence (SEQ ID NO: 4) of the 9th and the subsequent remainingnucleotides counted from the 5′-terminus of the above-describedhsa-miR-8053 (SEQ ID NO: 7) is fused to the 3′-terminal side of thenucleotide sequence as set forth in SEQ ID NO: 1. Similarly, apolynucleotide consisting of the nucleotide sequence as set forth in SEQID NO: 9 is an artificial polynucleotide in which the nucleotidesequence (SEQ ID NO: 5) of the 9th and the subsequent remainingnucleotides counted from the 5′-terminus of hsa-miR-4454 is fused to the3′-terminal side of the nucleotide sequence as set forth in SEQ ID NO:1, and hsa-miR-4454 is a miRNA having the nucleotide sequence as setforth in SEQ ID NO: 10 that is known as a cancer marker but is not knownto suppress tumor cells (Kojima M PLoS One. 10(2) (2015) “MicroRNAmarkers for the diagnosis of pancreatic and biliary-tract cancers”).

The polynucleotide of the present invention may have any structure, aslong as it exhibits the effects of treating and/or preventing cancer,and may have, for example, a single-stranded structure, adouble-stranded structure, or a multiple-stranded structure such as atriple- or more-stranded structure. The present polynucleotide haspreferably a single-stranded or double-stranded structure, and morepreferably a single-stranded structure.

The polynucleotide of the present invention may be RNA, DNA or RNA/DNA(chimera), as long as it exhibits the effects of treating and/orpreventing cancer. With regard to the polynucleotide of the presentinvention, when the entire or a part of the nucleotide sequencecorresponding to a sequence number shown in Sequence Listing is DNA, U(uracil) in the region corresponding to the DNA in the nucleotidesequence shown in Sequence Listing is replaced with T (thymine). Thepolynucleotide of the present invention is preferably RNA. Examples oftypes of RNA include mRNA, rRNA, non-coding RNA, siRNA, shRNA, snoRNA,snRNA, nkRNA (registered tradename), and PnkRNA (tradename), as well asthe above-mentioned miRNA. The RNA is preferably miRNA. The miRNAincludes synthetic miRNA that is what is called a mimic, as well asnaturally occurring miRNA.

The polynucleotide usable in the present invention can comprise at leastone modified nucleotide analog. The nucleotide analog can be placed, forexample, at the 5′ terminus, 3′ terminus, and/or inside of an RNAmolecule. In particular, by incorporation of the modified nucleotideanalog, the polynucleotide can be further stabilized.

The nucleotide analog is, for example, preferably a sugar- orbackbone-modified ribonucleotide, and more preferably a ribonucleotidehaving a modified nucleic acid base, and more specifically, aribonucleotide comprising a non-naturally occurring nucleic acid base.Examples of the non-naturally occurring nucleic acid base include, forexample, uridine or cytidine modified at position 5, such as5-methyluridine, 5-(2-amino)propyluridine, 5-methyl-2-thiouridine,5-bromouridine, or 6-azouridine; adenosine and guanosine modified atposition 8, such as 8-bromoguanosine, deazanucleotide, or7-deaza-adenosine; and O- and N-alkylated nucleotides;N6-methyladenosine; and universal bases.

A preferred sugar-modified ribonucleotide may be a ribonucleotide havinga substitution of a 2′-OH group in a sugar moiety with a group selectedfrom the group consisting of H, OR, halo, SH, SR, NH₂, NHR, NR₂ and CN,or a ribonucleotide comprising a 2′-O, 4′-C methylene bridge or anethylene bridge (for example, LNA or ENA), wherein R is C1 to C6 alkyl,alkenyl or alkynyl, and halo is F, Cl, Br or I. Further, the modifiedsugar moiety in the sugar modified ribonucleotide may be mannose,arabinose, glucopyranose, galactopyranose, 4′-thioribose or anothersugar; or a hetero ring or a carbon ring.

Examples of a preferred backbone-modified ribonucleotide includeribonucleotides having a substitution of a phosphoester group that bindsto an adjacent ribonucleotide, with, for example, aphosphothioate-modified group, boranophosphate, 3′-(or 5′)deoxy-3′-(or5′)aminophosphoramidate, hydrogen phosphonate, boranophosphate ester,phosphoramidate, alkyl, or aryl phosphonate and phosphotriester. Any ofthe above-mentioned modifications may be used in combination.

<Carrier to be Used Together with the Polynucleotide as ActiveIngredient>

The pharmaceutical composition for treating and/or preventing a cancerof the present invention may comprise a pharmaceutically acceptablecarrier, in addition to the polynucleotide of the present invention. Thepharmaceutically acceptable carrier is preferably a substance whichfacilitates the transport of the polynucleotide of the present inventionto target cells or tissues, does not stimulate a living body, and doesnot inhibit the activities and properties of the polynucleotide of thepresent invention, and it is also preferable that the carrier itselfdoes not induce the production of harmful antibodies to individuals towhich the composition is administered. The size of carrier is preferablya size which does not permeate normal blood vessel walls but canpermeate newborn blood vessels in cancer tissues. When a carrier is anapproximate spheroid, the diameter of the carrier may be preferably anano size of, for example, about 1 nm or more and less than 100 nm.

The carrier may encapsulate the polynucleotide of the present invention,or may movably bind to the polynucleotide of the present invention. Thephase “movably bind to” refers to the electronic interaction between thecarrier and one or more agents. The interaction is not limited, and maybe in the form of any chemical bonds, including covalent bond, polarcovalent bond, ionic bond, electrostatic bond, coordinate covalent bond,aromatic bond, hydrogen bond, and dipole or Van der Waals interaction.

The binding site of the polynucleotide of the present invention and thecarrier is preferably on the 5′ terminal side or on the 3′ terminalside, and more preferably on the 5′ terminal side.

Specific examples of the carrier include non-cationic polymer carriers,liposome carriers, dendrimer carriers, nano-material carriers,microparticle carriers, biostructural carriers, micelle carriers,polymer microparticles, and magnetic microparticles.

The non-cationic polymer carrier refers to a polymer that canencapsulate one or two or more agents therein, and/or can movably bindto such agent(s), and is, for example, anionic (i.e., negativelycharged) or an electronically neutral and cotton-like or branched. Thecarrier may be in the form of microparticles or nanoparticles, or mayalso be water-soluble or water-insoluble, or biodegradable ornon-biodegradable carrier. Preferred non-cationic polymer carriers areknown to those skilled in the art. The non-cationic polymer carrier mayinclude, for example, poly-L-glutamic acid (PGA), poly-(γ-L-glutamylglutamine) (PGGA), poly-(γ-L-aspartyl glutamine) (PGAA) orpoly-(lactide-co-glycolide) (PLGA); and a mixture of at least twopolymers.

The liposome carrier has a lipid double layer structure comprisinglipids attached to polar hydrophilic groups, which forms, in an aqueousmedium, a substantially closed structure which can encapsulate one ortwo or more agents and/or can movably bind to the agent(s). The liposomecarrier may comprise a single lipid double layer (i.e., unilamellar) ormay also comprise a concentric lipid double layer consisting of two orthree or more layers (i.e., multilamellar). The liposome carrier mayhave an approximate spherical or approximate elliptical shape. Preferredliposome carriers are known to those skilled in the art, and can beselected based on various properties such as the rigidity of the lipiddouble layer, the electronic charge of the lipid double layer and/or thecompatibility of one or both of the agents with the liposome carrier.The liposome carrier may comprise, for example, natural phospholipidssuch as egg phosphatidylcholine, egg phosphatidylethanolamine, soyphosphatidylcholine, lecithin and sphingomyelin, syntheticphosphatidylcholine, lysophosphatidylcholine, phosphatidylglycerol,phosphatidic acid, phosphatidyl ethanolamine, dioctadecylamideglycylspermine, dioleoylphosphatidylethanolamine,N-1-2,3-dioleyloxypropyl-N,N,N-trimethylammonium chloride,2,3-diolexyoloxy-N-2-sperminecarboxamidoethyl-N,N-dimethyl-1-propaneammoniumtrifluoroacetamide, phosphatidylserine, derivatives thereof, andPEGylated phospholipids.

The dendritic carrier refers to a dendrimer, a dendron, or derivativesthereof, which can encapsulate one or two or more agents, and/or canmovably bind to the agent(s). The dendrimer refers to a macromoleculehaving a core and a plurality of branch-structured shells spreading fromthe core. The dendron is a type of dendrimer having branches spreadingfrom a focal point. The dendritic carriers are commercially available orcan be synthesized by methods known to those skilled in the art. Thedendritic carrier may be at least partially hydrophobic or hydrophilic.The dendritic carrier may be cationic, or electronically neutral, oranionic. The dendritic carrier may comprise a core molecule, andexamples thereof include alkyl diamines such as ethylenediamine,1,4-diaminobutane, 1,6-diaminohexane and 1,12-diaminodecane; amines suchas ammonia; alkylimines such as cystamine and polyethyleneimine (PEI);and chlorinated phosphorus molecules such as cyclotriphosphazene andthiophosphoryl. The dendritic carrier may comprise alkylimines such aspolypropyleneimine (PPI), tertiary amines such as polyamideamine(PAMAM), polyamino acids such as polylysine, and/or phenoxymethyl(methylhydrazono) (PMMH).

The nano-material carrier refers to a material having the longestdimension ranging from about 1 nm to about 100 nm, which can encapsulateone or two or more agents and/or can movably bind to the agent(s).Preferred nano-material carriers are known to those skilled in the art,and examples of the nano-material carrier may include nanoparticle,nanopowder, nanocluster, nanocrystal, nanosphere, nanofiber, nanotube,nanocluster, nanocrystal, nanosphere, nanofiber, nanotube, nanogel,and/or nanorod. The examples of the substance constituting thenano-material carrier include poly-(lactide-co-glycolide) (PLGA),polyalkylcyanoacrylate (PACA), polyepsilon-caprolactone (PCL),polylactic acid (PLA), polyethyleneglycol (PEG), poly-N-vinylcaprolactamsodium acrylate, poly-N-isopropylacrylamide, and polyvinyl acetate.Further, in some aspects, the nano-material carrier may be fullerene,and the fulleren may include spherical fullerenes (e.g., C60), carbonnanotubes, and fullerene derivatives.

The microparticle carrier refers to a particle having the longestdimension ranging from about 100 nm to about 100 μm. The microparticlemay have any shapes and any forms. Examples of the substanceconstituting the microparticle carrier includepoly-(lactide-co-glycolide) (PLGA), polyalkylcyanoacrylate (PACA),polyepsilon-caprolactone (PCL), polylactic acid (PLA), PLGA, andpolyethyleneglycol (PEG).

The biostructural carrier refers to a polymer or a compound in which alarge number of units in the biostructural carrier are amino acidsand/or saccharides, and which can encapsulate one or two or more agentsand/or movably bind to the agent(s). Preferred biostructural carriersare known to those skilled in the art, and may comprise any of sugars,monosaccharides, oligosaccharides, polysaccharides, cyclicpolysaccharides, non-cyclic polysaccharides, linear polysaccharides,branched polysaccharides, amino acids, proteins, and peptides; andsemisynthesized derivatives thereof. The biostructural carrier maycomprise any of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, methylβ-cyclodextrin, dimethyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin,hydroxypropyl-β-cyclodextrin, sulfobutyl ether-β-cyclodextrin,tri-O-methyl-β-cyclodextrin, glucosyl-β-cyclodextrin, β1,3D glucan, β1,6glucan, C-reactive protein, conalbumin, lactalbumin, ovalbumin,parvalbumin, serum albumin, technetium TC99m aggregated albumin, humanserum albumin (HSA), bovine serum albumin (BSA), recombinant human serumalbumin (rHSA), glucose (dextrose), fructose, galactose, xylose, ribose,sucrose, cellulose, cyclodextrin, and starch.

The micelle carrier has a micelle structure formed by lipids, such asany fat-soluble (or lipophilic) molecule, oil, wax, sterol,monoglyceride, diglyceride, triglyceride, phospholipid, etc. The micellecarrier may comprise any of polyalkylene glycols such as polyethyleneglycol (PEG); polyamino acids such as polyaspartic acid and polyglutamicacid (PGA); poly-(γ-L-glutamyl glutamine) (PGGA), polyphenyleneoxide(PPO), poly(ε-caprolactone) (PCL), poly-(lactide-co-glycolide) (PLGA),and a diblock copolymer.

Further, the carrier may also be a conjugate, and may comprise anucleotide linker, a non-nucleotide linker or anucleotide/non-nucleotide complex linker, which links a sense regionwith an antisense region of a nucleic acid; polyethylene glycol; humanserum albumin; and a ligand for a cell receptor, capable of inducing thecellular uptake. Additionally, the nucleotide linker may be a linkerhaving 2 or more nucleotides in length, or may also be a nucleic acidaptamer.

The pharmaceutical composition for treating and/or preventing a cancercomprising the polynucleotide of the present invention may furthercomprise at least one substance selected from pharmaceuticallyacceptable excipients, pharmaceutical carriers and diluents. The presentpolynucleotide can be formulated into any dosage forms includingparenteral dosage forms such as injection dosage forms, or formssuitable for intrarectal, intranasal, local, subcutaneous, vaginal orother parenteral administration, or oral dosage forms such as pills,capsules, granules or tablets, or forms suitable for inhalation orinfusion administration, with further adding a diluent, dispersant,surfactant, binder, lubricant and/or a mixture thereof to thepolynucleotide.

When the polynucleotide of the present invention is used as a liquidpreparation, the carrier is preferably sterilized and suitable for aliving body, and further, other common additives such as an antioxidant,a buffer solution and a bacteriostatic agent may also be added.Preferred additives include, but are not limited to, macromolecules thatare large and slowly metabolized, such as proteins, polysaccharides,polylactic acid, polyglycolic acid, polymeric amino acids, amino acidcopolymer, lipid aggregate, hydrogel, inactivated virus particle, andcollagens. Further, a liquid preparation comprising the polynucleotideof the present invention may comprise liquids such as water, saline,sterilized water, Ringer's solution, buffered saline, albumin injectionsolution, dextrose solution, maltodextrin solution, glycerol or ethanol,and may also comprise auxiliary substances such as moistening agent,emulsifying agent, a pH buffering substance and the like.

In the context of the present invention, the term “administration” meansintroduction of the polynucleotide of the present invention or apharmaceutical composition for treating and/or preventing a cancercomprising the polynucleotide of the present invention as an activeingredient, to a patient by any suitable method. The administrationincludes the delivery of the polynucleotide of the present invention bya viral or non-viral technique, and transplantation of cells whichexpress the polynucleotide of the present invention.

The administration can be carried out via various oral or parenteraladministration routes, as long as the polynucleotide can reach a targettissue. For example, the administration can be carried out by anintraoral, intrarectal, local, intravenous, intraperitoneal,intramuscular, intraarterial, transdermal, intranasal, inhalation,intraocular, or intradermal route.

The dose varies depending on the purpose of administration, anadministration method, type and size of a tumor, characteristics (sex,age, body weight, etc.) of a person to be administered (i.e., asubject). Typically, with regard to the dose, a drug is administered ata lower level and is then increased until the intended effect isachieved. A preferred dose of the polynucleotide of the presentinvention may range, but not limited to, for example, from 1 pmol to 100nmol per kg of body weight; or from 0.001 to 0.25 mg per kg of bodyweight, or from 0.01 to 20 μg per kg of body weight, or from 0.10 to 5μg per kg of body weight. Such doses are administered preferably 1 to 10times, and more preferably 5 to 10 times.

<Suppression of Cancers by the Polynucleotide>

The polynucleotide of the present invention may be provided in a form ofa polynucleotide introduced into cells. The phrase “introduced intocells” means the entry of a foreign polynucleotide into cells bytransfection or transduction. The transfection refers to, for example,calcium phosphate-DNA co-precipitation, DEAE-dextran-mediatedtransfection, polybrene-mediated transfection, electroporation,microinjection, liposome fusion, Lipofectamine transfection, andprotoplast fusion. The transduction means the transfer of a gene intoother cells by means of infection using a virus or a viral vectorparticle (e.g., a vector based on adenovirus, adeno-associated virus,Sendai virus, or retrovirus (lentivirus, etc.)) or using a plasmidvector. The vector can comprise necessary elements (e.g., a promoter,etc.) for enabling the expression of the polynucleotide of the presentinvention, and can be prepared by known techniques (e.g., Sambrook andRussell, Molecular Cloning A Laboratory Manual (4^(th) Ed., 2001), ColdSpring Harbor Laboratory Press, JP Patent Publication No. 2016-153403 A,JP Patent Publication No. 2016-025853 A). The cells into which thepolynucleotide of the present invention has been introduced by suchmethods, can express the polynucleotide of the present invention at ahigh level. Thus, the cells can be utilized as a cell-therapeutic agentfor suppressing the growth of cancer when transplanted into cancertissues.

<Type of Cancer>

The terms “tumor” and “cancer” are used in the context of the presentinvention to mean malignant neoplasms and are used interchangeably. Thecancer to be targeted includes, but is not particularly limited to, asolid cancer. Specific examples of the cancer to be targeted includecancers and cancer cells developed in, or derived from, bladder, bone,bone marrow, brain, breast, colon/rectum, esophagus, digestive tract,gum, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate,skin, stomach, testis, tongue, blood or uterus. Preferred examples ofthe cancer include breast cancer, kidney cancer, pancreatic cancer,colorectal cancer, lung cancer, brain tumor, stomach cancer, cervicalcancer, uterine cancer, ovarian cancer, prostate cancer, bladder cancer,esophagus cancer, liver cancer, fibrosarcoma, mast cell tumor, andmelanoma. Specific examples of these cancers include, but are notlimited to, mammary gland cancer, complex mammary gland carcinoma,mammary gland malignant mixed tumor, intraductal papillaryadenocarcinoma, lung adenocarcinoma, squamous cell carcinoma, small cellcarcinoma, large cell carcinoma, glioma which is a neuroepithelialtissue tumor, ependymoma, neurocytoma, embryonal neuroectodermal tumor,schwannoma, neurofibroma, meningioma, chronic lymphocytic leukemia,lymphoma, GI lymphoma, digestive lymphoma, small to medium celllymphoma, cecal cancer, ascending colon cancer, descending colon cancer,transverse colon cancer, sigmoid colon cancer, rectal cancer, epithelialovarian cancer, germ cell tumor, stromal cell tumor, pancreatic ductalcancer, invasive pancreatic ductal cancer, adenocarcinomas of pancreaticcancer, acinic cell carcinoma, adenosquamous carcinoma, giant celltumor, intraductal papillary mucinous neoplasm, mucinouscystadenocarcinoma, pancreatoblastoma, serous cystadenocarcinoma, solidpapillary cancer, gastrinoma, glucagonoma, insulinoma, multipleendocrine adenomatosis, non-functional islet cell tumor,somatostatinoma, and VIPoma.

Moreover, preferred subjects to be targeted in the present invention aremammals, including primates such as human, livestock such as cow, pig,sheep and horse, companion animals such as dog and cat, and mammals in azoo. Among others, human is preferable.

In the present invention, the polynucleotide of the present invention,or the pharmaceutical composition for treating and/or preventing acancer according to the present invention, can be administered to thesubject for treating and/or preventing a cancer.

<Type of Antitumor Agent>

In the present invention, a drug referred to as a “combination drug”using a pharmaceutical composition for treating and/or preventing acancer comprising, as an active ingredient, the polynucleotide of thepresent invention, in combination with another (typically, known)antitumor agent, or with a pharmaceutical composition comprising anotherantitumor agent can be administered to a subject in combination, andthereby preferably increasing the antitumor effects. The pharmaceuticalcomposition for treating and/or preventing a cancer according to thepresent invention and another antitumor agent (or a pharmaceuticalcomposition comprising another antitumor agent) can be administered to asubject, simultaneously or separately. In the case of the separateadministration, either the pharmaceutical composition or the antitumoragent may be administered earlier or later, and the dosing interval,doses, administration routes and the numbers of doses can be determinedappropriately by a medical specialist. Another dosage form of the drugto be simultaneously administered includes, for example, apharmaceutical composition also referred to as a “mixed drug” preparedby mixing and formulating the pharmaceutical composition for treatingand/or preventing a cancer according to the present invention with anantitumor agent in a pharmaceutically acceptable carrier (or medium).

Examples of the antitumor agent include the following antitumor agentsknown in literatures or the like.

Examples of the antitumor agent as an alkylating agent such as Thiotepaand cyclophosphamide include: alkyl sulfonates such as (i.e.,“including”) busulfan, improsulfan, and piposulfan; aziridines such asbenzodopa, carboquone, meturedopa, and uredopa; ethyleneimines such asaltretamine, triethyleneamine, triethylenephosphoramide,triethylenethiophosphoramide, and trimethylolamine; acetogenins such asbullatacin and bullatacinone; camptothecin; bryostatin; callystatin;cryptophycin 1, and cryptophycin 8; dolastatin; duocarmycin;eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide, andestramustine; ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, temozolomide, novembichin; fenesterin,prednimustine, trofosfamide, uracil mustard; and nitrosoureas such asbendamustine, carmustine, chlorozotocin, streptozocin, fotemustine,lomustine, nimustine, and ranimnustine.

Examples of the antitumor agent as an anticancer antibiotic includecalicheamicin, dynemicin, clodronate, esperamicin, aclacinomycin,actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabicin,carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin,detorubicin, 6-diazo-5-oxo-L-norleucine, adriamycin (doxorubicin),bleomycin, aclarbicin, amrubicin, epirubicin, esorubicin, idarubicin,marcellomycin, mitomycin C, mycophenolic acid, nogalamycin, olivomycin,peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin,streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, andzorubicin.

Examples of the antitumor agent as an antimetabolite include: folic acidanalogs such as denopterin, pteropterin, methotrexate, trimetrexate, andpemetrexed; purine analogs such as fludarabine, 6-mercaptopurine,thiamiprine, thioguanine, cladribine, and clofarabine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine,trifluridine, capecitabine, 5-FU, gemcitabine, S-1, and tegafur; andhydroxycarbamide and nelarabine.

Examples of the antitumor agent as a hormone preparation includeanastrozole, bicalutamide, degarelix, estramustine, exemestane,flutamide, fulvestrant, goserelin, letrozole, leuplin,medroxyprogesterone, mepitiostane, octreotide, tamoxifen, andtoremifene, and, for example, androgen preparations such as calusterone,drostanolone propionate, epitiostanol, mepitiostane, testolactone, andenzalutamide; antiadrenal preparations such as aminoglutethimide,mitotane, and trilostane; and frolinic acid, aceglatone, aldophosphamideglycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil,bisantrene, edatraxate, defofamine, demecolcine, diaziquone,elformithine, elliptinium acetate, epothilone, etoglucid, lenthinan,lonidamine, maytansine, ansamitocine, abiraterone, mitoguazone,mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet,pirarubicin, losoxantrone, podophyllic acid, 2-ethyl hydrazide,procarbazine, razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonicacid, triaziquone, roridin A, anguidine, urethane, vindesine,dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman,gacytosine, arabinoside, BCG, Krestin, and picibanil.

Examples of the antitumor agent as another anticancer agent such asthose derived from plants include docetaxel, etoposide, teniposide,irinotecan, nogitecan, paclitaxel, cabazitaxel, vinblastine,vincristine, vindesine, vinorelbine, carboplatin, cisplatin,dacarbazine, eribulin, L-asparaginase, miriplatin, mitoxantrone,nedaplatin, oxaliplatin, pentostatin, procarbazine, arsenic trioxide,sobuzoxane, tamibarotene, mitoxantrone, novantrone, edatrexate,ibandronate, topoisomerase inhibitor, difluoromethylornithine (DMFO),and retinoic acid.

Examples of the antitumor agent as a molecular target drug includeafatinib, axitinib, alectinib, bevacizumab, cetuximab, crizotinib,erlotinib, everolimus, gefitinib, lapatinib, ramucirumab, panitumumab,pazopanib, pertuzumab, nivolumab, regorafenib, lenvatinib, sorafenib,sunitinib, temsirolimus, trastuzumab, and pharmaceutically acceptablesalts or derivatives thereof.

Further, radioisotopes such as ²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re,¹⁵³SM, ²¹²Bi, ³²P, ¹⁷⁵Lu, ¹⁷⁶Lu, ⁸⁹Sr, ²²³Ra and ¹⁶¹Tb, which are knownin literatures and the like, may also be used as antitumor agents. Theradioisotopes are desirably those effective for treating and diagnosingtumors, and such radioisotopes may also be composed in thepharmaceutical composition for treating and/or preventing a canceraccording to the present invention.

<Treatment and Prevention Methods>

The present invention further provides a method for treating and/orpreventing a cancer in a subject who suffers (or has suffered) from acancer, comprising administering the pharmaceutical composition fortreating and/or preventing a cancer according to the present invention,or a combination drug comprising the pharmaceutical composition fortreating and/or preventing a cancer according to the present inventionand the above-mentioned another antitumor agent (or a pharmaceuticalcomposition comprising the antitumor agent), to the subject.

The terms “treating a cancer” and “antitumor effects” used herein referto the effects on cancer cells or tumors, compared with a negativecontrol which is not treated with the polynucleotide of the presentinvention or the pharmaceutical composition for treating and/orpreventing a cancer according to the present invention, wherein theeffects include not only complete inhibition of the growth of cancercells and regression or disappearance of tumors, but also delay in theincrease of cancer cells (i.e., reduction in the increment of cancercells) or delay in the tumor growth compared with a negative controlwhich is not treated with the polynucleotide of the present invention orthe pharmaceutical composition for treating and/or preventing a canceraccording to the present invention.

The term “prevention” used herein also includes prevention of cancerrecurrence for reducing a risk of recurrence after cancer treatment bycancer therapy such as surgery, chemotherapy, radiotherapy, orimmunotherapy.

The above descriptions of pharmaceutical composition, combination drug,polynucleotide as an active ingredient, dose, usage, dosage form,cancers to be targeted, and the like also apply to the methods of thissection.

EXAMPLES

The present invention will be further specifically described inreference to the following Examples. However, these examples are notintended to limit the scope of the present invention.

[Example 1] Effectiveness of Synthetic RNAs on Pancreatic Cancer Cells

A synthetic RNA having (i.e., consisting of; the same applies regardingsequences in the present description) the same nucleotide sequence ashsa-miR-4450 as set forth in SEQ ID NO: 6, and a synthetic RNA havingthe same nucleotide sequence as hsa-miR-8053 as set forth in SEQ ID NO:7, were each evaluated for the effectiveness on pancreatic cancer cells.

A Panc-1 cell line (ATCC® CRL-1469™) as pancreatic cancer cells wasseeded in a DMEM medium (Nacalai Tesque, Japan) supplemented with 10%FBS, and was then cultured under conditions of 37° C. and 5% CO₂. Panc-1pancreatic cancer cells were seeded at 6×10³ cells per well in 96-wellplates. Thereafter, RNA synthetic products (Thermo Fisher ScientificInc., mirVana™ miRNA Mimics) having the nucleotide sequences as setforth in SEQ ID NOs: 6 and 7 or a negative control oligo (Thermo FisherScientific Inc., mirVana™ miRNA Mimic, Negative Control) were each addedat a concentration of 30 nM and introduced into the pancreatic cancercells using Lipofectamine RNAiMAX (Thermo Fisher Scientific Inc.). Theculture medium was exchanged 24 hours after the gene introduction, andthe number of cells was measured for 5 days. The number of cells wasdetermined by measuring ATP activity using the Celtiter-glo (PromegaCorporation) reagent, and the measurement value was used as an indicatorof the number of surviving cells. The results are shown in FIG. 1. Theevaluation test was carried out at n=3. The graph of FIG. 1 indicatesthe mean±standard deviation of the viability (%) of the pancreaticcancer cells compared with the negative control.

As a result, pancreatic cancer cells into which the RNA syntheticproducts having the nucleotide sequences as set forth in SEQ ID NOs: 6and 7 had been introduced, were found to have cell viability of 31% and75%, respectively, compared with pancreatic cancer cells into which thenegative control oligo had been introduced.

[Example 2] Effectiveness of Synthetic RNAs on Breast Cancer Cells

The synthetic RNA having the same nucleotide sequence as hsa-miR-4450 asset forth in SEQ ID NO: 6 and the synthetic RNA having the samenucleotide sequence as hsa-miR-8053 as set forth in SEQ ID NO: 7 wereeach evaluated for the effectiveness on breast cancer cells.

An MCF-7 cell line (ATCC® HTB-22™) as breast cancer cells was seeded inan RPMI medium (Nacalai Tesque, Japan) supplemented with 10% FBS, andwas then cultured under conditions of 37° C. and 5% CO₂. MCF-7 breastcancer cells were seeded at 6×10³ cells per well in 96-well plates.Thereafter, RNA synthetic products (Thermo Fisher Scientific Inc.,mirVana™ miRNA Mimics) having the nucleotide sequences as set forth inSEQ ID NOs: 6 and 7 or the negative control oligo (Thermo FisherScientific Inc., mirVana™ miRNA Mimic, Negative Control) were each addedat a concentration of 30 nM and introduced into the breast cancer cellsusing Lipofectamine RNAiMAX (Thermo Fisher Scientific Inc.). The culturemedium was exchanged 24 hours after the gene introduction, and thenumber of cells was measured for 5 days. The number of cells wasdetermined by measuring ATP activity using the Celtiter-glo (PromegaCorporation) reagent, and the measurement value was used as an indicatorof the number of surviving cells. The results are shown in FIG. 2. Theevaluation test was carried out at n=3. The graph of FIG. 2 indicatesthe mean±standard deviation of the viability (%) of the breast cancercells compared with the negative control. As a result, breast cancercells into which the RNA synthetic products having the nucleotidesequences as set forth in SEQ ID NOs: 6 and 7 had been introduced, werefound to have cell viability of 66% and 85%, respectively, compared withbreast cancer cells into which the negative control oligo had beenintroduced.

[Example 3] Effectiveness of Synthetic RNAs on Lung Cancer Cells

The synthetic RNA having the same nucleotide sequence as hsa-miR-4450 asset forth in SEQ ID NO: 6 and the synthetic RNA having the samenucleotide sequence as hsa-miR-8053 as set forth in SEQ ID NO: 7 wereeach evaluated for the effectiveness on lung cancer cells.

An A549 cell line (ATCC® CCL-185™) as lung cancer cells was seeded in anRPMI medium (Nacalai Tesque, Japan) supplemented with 10% FBS, and wasthen cultured under conditions of 37° C. and 5% CO₂. A549 lung cancercells were seeded at 3×10³ cells per well in 96-well plates. Thereafter,RNA synthetic products (Thermo Fisher Scientific Inc., mirVana™ miRNAMimics) having the nucleotide sequences as set forth in SEQ ID NOs: 6and 7 or the negative control oligo (Thermo Fisher Scientific Inc.,mirVana™ miRNA Mimic, Negative Control) were each added at aconcentration of 30 nM and introduced into the lung cancer cells usingLipofectamine RNAiMAX (Thermo Fisher Scientific Inc.). The culturemedium was exchanged 24 hours after the gene introduction, and thenumber of cells was measured for 5 days. The number of cells wasdetermined by measuring ATP activity using the Celtiter-glo (PromegaCorporation) reagent, and the measurement value was used as an indicatorof the number of surviving cells. The results are shown in FIG. 3. Theevaluation test was carried out at n=3. The graph of FIG. 3 indicatesthe mean±standard deviation of the viability (%) of the lung cancercells compared with the negative control. As a result, lung cancer cellsinto which the RNA synthetic products having the nucleotide sequences asset forth in SEQ ID NOs: 6 and 7 had been introduced, were found to havecell viability of 44% and 84%, respectively, compared with lung cancercells into which the negative control oligo had been introduced.

[Example 4] Effectiveness of Synthetic RNAs on Stomach Cancer Cells

The synthetic RNA having the same nucleotide sequence as hsa-miR-4450 asset forth in SEQ ID NO: 6 and the synthetic RNA having the samenucleotide sequence as hsa-miR-8053 as set forth in SEQ ID NO: 7 wereeach evaluated for the effectiveness on stomach cancer cells.

An NC1-N87 cell line (ATCC® CRL-5822™) as stomach cancer cells wasseeded in an RPMI medium (Nacalai Tesque, Japan) supplemented with 10%FBS, and was then cultured under conditions of 37° C. and 5% CO₂. Thestomach cancer cells were seeded at 6×10³ cells per well in 96-wellplates. Thereafter, RNA synthetic products (Thermo Fisher ScientificInc., mirVana™ miRNA Mimics) having the nucleotide sequences as setforth in SEQ ID NOs: 6 and 7 or the negative control oligo (ThermoFisher Scientific Inc., mirVana™ miRNA Mimic, Negative Control) wereeach added at a concentration of 30 nM and introduced into the stomachcancer cells using Lipofectamine RNAiMAX (Thermo Fisher ScientificInc.). The culture medium was exchanged 24 hours after the geneintroduction, and the number of cells was measured for 5 days. Thenumber of cells was determined by measuring ATP activity using theCeltiter-glo (Promega Corporation) reagent, and the measurement valuewas used as an indicator of the number of surviving cells. The resultsare shown in FIG. 4. The evaluation test was carried out at n=3. Thegraph of FIG. 4 indicates the mean±standard deviation of the viability(%) of the stomach cancer cells compared with the negative control. As aresult, stomach cancer cells into which the RNA synthetic productshaving the nucleotide sequences as set forth in SEQ ID NOs: 6 and 7 hadbeen introduced, were found to have cell viability of 57% and 68%,respectively, compared with stomach cancer cells into which the negativecontrol oligo had been introduced.

[Example 5] Effectiveness of Synthetic RNAs on Liver Cancer Cells

The synthetic RNA having the same nucleotide sequence as hsa-miR-4450 asset forth in SEQ ID NO: 6 and the synthetic RNA having the samenucleotide sequence as hsa-miR-8053 as set forth in SEQ ID NO: 7 wereeach evaluated for the effectiveness on liver cancer cells.

A HepG2 cell line (ATCC® HB-8065™) as liver cancer cells was seeded inan RPMI medium (Nacalai Tesque, Japan) supplemented with 10% FBS, andwas then cultured under conditions of 37° C. and 5% CO₂. The livercancer cells were seeded at 6×10³ cells per well in 96-well plates.Thereafter, RNA synthetic products (Thermo Fisher Scientific Inc.,mirVana™ miRNA Mimics) having the nucleotide sequences as set forth inSEQ ID NOs: 6 and 7 or the negative control oligo (Thermo FisherScientific Inc., mirVana™ miRNA Mimic, Negative Control) were each addedat a concentration of 30 nM and introduced into the liver cancer cellsusing Lipofectamine RNAiMAX (Thermo Fisher Scientific Inc.). The culturemedium was exchanged 24 hours after the gene introduction, and thenumber of cells was measured for 5 days. The number of cells wasdetermined by measuring ATP activity using the Celtiter-glo (PromegaCorporation) reagent, and the measurement value was used as an indicatorof the number of surviving cells. The results are shown in FIG. 5. Theevaluation test was carried out at n=3. The graph of FIG. 5 indicatesthe mean±standard deviation of the viability (%) of the liver cancercells compared with the negative control. As a result, liver cancercells into which the RNA synthetic products having the nucleotidesequences as set forth in SEQ ID NOs: 6 and 7 had been introduced, werefound to have cell viability of 71% and 81%, respectively, compared withliver cancer cells into which the negative control oligo had beenintroduced.

[Example 6] Effectiveness of Synthetic RNAs on Colorectal Cancer Cells(1)

The synthetic RNA having the same nucleotide sequence as hsa-miR-4450 asset forth in SEQ ID NO: 6 and the synthetic RNA having the samenucleotide sequence as hsa-miR-8053 as set forth in SEQ ID NO: 7 wereeach evaluated for the effectiveness on colorectal cancer cells.

An HCT116 cell line (ATCC® CCL-247™) as colorectal cancer cells wasseeded in a McCoy's medium (Nacalai Tesque, Japan) supplemented with 10%FBS, and was then cultured under conditions of 37° C. and 5% CO₂. HCT116colorectal cancer cells were seeded at 6×10³ cells per well in 96-wellplates. Thereafter, RNA synthetic products (Thermo Fisher ScientificInc., mirVana™ miRNA Mimics) having the nucleotide sequences as setforth in SEQ ID NOs: 6 and 7 or the negative control oligo (ThermoFisher Scientific Inc., mirVana™ miRNA Mimic, Negative Control) wereeach added at a concentration of 30 nM and introduced into thecolorectal cancer cells using Lipofectamine RNAiMAX (Thermo FisherScientific Inc.). The culture medium was exchanged 24 hours after thegene introduction, and the number of cells was measured for 5 days. Thenumber of cells was determined by measuring ATP activity using theCeltiter-glo (Promega Corporation) reagent, and the measurement valuewas used as an indicator of the number of surviving cells. The resultsare shown in FIG. 6. The evaluation test was carried out at n=3. Thegraph of FIG. 6 indicates the mean±standard deviation of the viability(%) of the colorectal cancer cells compared with the negative control.As a result, colorectal cancer cells into which the RNA syntheticproducts having the nucleotide sequences as set forth in SEQ ID NOs: 6and 7 had been introduced, were found to have cell viability of 4% and46%, respectively, compared with colorectal cancer cells into which thenegative control oligo had been introduced.

[Example 7] Effectiveness of Synthetic RNAs on Colorectal Cancer Cells(2)

A synthetic RNA having the nucleotide sequence as set forth in SEQ IDNO: 8 (the nucleotide sequence in which SEQ ID NO: 4, namely, thenucleotide sequence ranging from positions 9 to 24 counted from the5′-terminus of SEQ ID NO: 7, is added to the 3′-terminus of SEQ ID NO:1), a synthetic RNA having the nucleotide sequence as set forth in SEQID NO: 9 (the nucleotide sequence in which the nucleotide sequence ofSEQ ID NO: 5 that ranges from positions 9 to 20 counted from the5′-terminus of hsa-miR-4454 as set forth in SEQ ID NO: 10, is added tothe 3′-terminus of SEQ ID NO: 1, namely, the nucleotide sequence rangingfrom positions 1 to 8 counted from the 5′-terminus of SEQ ID NO: 6), anda synthetic RNA having the same nucleotide sequence as hsa-miR-4454 asset forth in SEQ ID NO: 10 known as a cancer marker (Thermo FisherScientific Inc., mirVana™ miRNA Mimics) were each evaluated for theeffectiveness on colorectal cancer, by the same method as that describedin Example 6.

As a result, colorectal cancer cells into which the RNA syntheticproducts having the nucleotide sequences as set forth in SEQ ID NOs: 8and 9 had been introduced, and colorectal cancer cells into which theRNA synthetic product having the nucleotide sequence as set forth in SEQID NO: 6 had been introduced (Example 6), were found to have cellviability of 20% or less. In contrast, the cell viability of colorectalcancer cells into which the RNA synthetic product having the nucleotidesequence as set forth in SEQ ID NO: 10 had been introduced, was 100%,and thus, the RNA synthetic product having the nucleotide sequence asset forth in SEQ ID NO: 10 was hardly effective. The results are shownin FIG. 7.

All of the synthetic RNAs consisting of the nucleotide sequences as setforth in SEQ ID NOs: 6, 8 and 9, each of which comprises the nucleotidesequence as set forth in SEQ ID NO: 1 at the 5′-terminus thereof,significantly reduced the cell viability of colorectal cancer cells. Thesynthetic RNA having the nucleotide sequence as set forth in SEQ ID NO:10, which has the same sequence on the 3′-terminal side as that of SEQID NO: 9, did not exhibit effectiveness. Accordingly, it was shown thatthe nucleotide sequence on the 5′-terminal side (in particular, thenucleotide sequence ranging from positions 1 to 8 counted from the5′-terminus) is important for anticancer effects.

[Comparative Example 1] Effectiveness of Synthetic RNAs on ColorectalCancer Cells

A synthetic RNA having the same nucleotide sequence as hsa-miR-575 asset forth in SEQ ID NO: 11 known as a cancer marker and a synthetic RNAhaving the same nucleotide sequence as hsa-miR-1321 as set forth in SEQID NO: 12, which is known to be associated with blood cancer (ThermoFisher Scientific Inc., mirVana™ miRNA Mimics), were each evaluated forthe effectiveness on colorectal cancer, by the same method as thatdescribed in Example 6.

As a result, colorectal cancer cells into which the RNA syntheticproducts having the nucleotide sequences as set forth in SEQ ID NOs: 6and 7 had been introduced, were found to have cell viability of 50% orless. In contrast, colorectal cancer cells into which the RNA syntheticproducts having the nucleotide sequences as set forth in SEQ ID NOs: 11and 12 had been introduced, were found to have cell viability of 96% and93%, respectively. The results are shown in FIG. 8.

[Comparative Example 2] Influence of Synthetic RNA on Normal Cells

The influence of the synthetic RNA having the nucleotide sequence as setforth in SEQ ID NO: 6 (Thermo Fisher Scientific Inc., mirVana™ miRNAMimics) on mammary epithelial cells, which are normal cells, wasevaluated.

A 184B5 cell line (ATCC® CRL-8799™) as mammary epithelial cells wasseeded in an MEBM medium (Lonza) supplemented with BPE, hydrocortisone,hEGF and insulin, and was then cultured under conditions of 37° C. and5% CO₂. The cells were seeded at 6×10³ cells per well in 96-well plates.Thereafter, the RNA synthetic product having the nucleotide sequence asset forth in SEQ ID NO: 6 or the negative control oligo (Thermo FisherScientific Inc., mirVana™ miRNA Mimic, Negative Control) was added at aconcentration of 3 nM and introduced into the cells using LipofectamineRNAiMAX (Thermo Fisher Scientific Inc.). The culture medium wasexchanged 24 hours after the gene introduction, and the number of cellswas measured for 5 days. The number of cells was determined by measuringATP activity using the Celtiter-glo (Promega Corporation) reagent, andthe measurement value was used as an indicator of the number ofsurviving cells.

The results are shown in FIG. 9. The evaluation test was carried out atn=3. The graph of FIG. 9 indicates the mean±standard deviation of theviability (%) of the pancreatic cancer cells compared with the negativecontrol. As a result, mammary epithelial cells into which the RNAsynthetic product (3 nM) having the nucleotide sequence as set forth inSEQ ID NO: 6 had been introduced, was found to have cell viability of93%, and thus, the influence of the synthetic RNA on normal cells wasnot observed (FIG. 9).

[Example 8] Effectiveness of Synthetic RNA on Cancer-Bearing MouseModels

Using cancer-bearing mice into which a human-derived cancer cell linehas been transplanted, the antitumor effects of the synthetic RNA havingthe same nucleotide sequence as hsa-miR-4450 as set forth in SEQ ID NO:6 were examined.

A human colorectal cancer cell line HCT116 (ATCC® CCL-247™) wassubcutaneously transplanted into the back of six Balb/c nude mice(Charles River Japan Inc.) at 5×10⁶ cells per mouse, and the tumor wasgrown until its diameter reached about 5 mm. To each of the sixcancer-bearing mice, a mixed solution of the synthetic RNA having thenucleotide sequence as set forth in SEQ ID NO: 6 (Thermo FisherScientific Inc., mirVana™ miRNA Mimics) or the negative control oligo(Thermo Fisher Scientific Inc., mirVana™ miRNA Mimic, Negative Control)(2 nmol per mouse) and 50 μl of 0.5% AteloGene® Local Use (KOKEN CO.,LTD.) was subcutaneously administered around the tumor. Subsequently,the mixed solution of the synthetic RNA and 0.5% AteloGene® Local Usewas subcutaneously administered at the same dose every 2 days, 3 timesin total around the tumor of the cancer-bearing mouse, and the size ofthe tumor was measured once every two days. The size of the tumor wascalculated as a volume, using the formula: 0.5×(long diameter×shortdiameter×short diameter).

As a result, on Day 13 after the initial administration, the tumorvolume ratio of the test group to which the synthetic RNA having thesame nucleotide sequence as SEQ ID NO: 6 had been administered was 48%,relative to the tumor volume of the negative control group (to which thenegative control oligo had been administered) set at 100% (FIG. 10A). Inaddition, changes in the tumor volume for 13 days after theadministration of the synthetic RNA to the cancer-bearing mice are shownin FIG. 10B.

From these results, it was shown that the synthetic RNA having thenucleotide sequence as SEQ ID NO: 6 exhibits antitumor effects in vivoon cancer cells.

INDUSTRIAL APPLICABILITY

The pharmaceutical composition for treating a cancer according to thepresent invention is useful for treating and/or preventing a cancer.

All publications, patents, and patent applications cited in the presentdescription are incorporated herein by reference in their entirety.

The invention claimed is:
 1. A method for treating a cancer in a subjectwho suffers or has suffered from the cancer, comprising administering apharmaceutical composition for treating a cancer to the subject, whereinthe pharmaceutical composition comprises, as an active ingredient, apolynucleotide comprising the nucleotide sequence as set forth in SEQ IDNO:1 or 2 and, wherein the polynucleotide further comprises a nucleotidesequence of the following (a) or (b) on the 3′ terminal side of thenucleotide sequence asset forth in SEQ ID NO:1 or 2: (a) the nucleotidesequence as set forth in any one of SEQ ID NOs: 3 to 5, or (b) anucleotide sequence comprising a deletion, substitution or insertion of1 to 3 nucleotides in the nucleotide sequence as set forth in any one ofSEQ ID NOs: 3 to 5, and wherein the cancer is pancreatic cancer, breastcancer, lung cancer, stomach cancer, liver cancer, or colorectal cancer.2. The method according to claim 1, wherein said pharmaceuticalcomposition is administered in combination with an antitumor agent. 3.The method according to claim 1, wherein the polynucleotide is 8 to 60nucleotides in length.
 4. The method according to claim 1, wherein thepolynucleotide comprises the nucleotide sequence as set forth in any oneof SEQ ID NOs: 6 to
 9. 5. The method according to claim 1, wherein thepolynucleotide is single stranded or double stranded.
 6. The methodaccording to claim 1, wherein the polynucleotide is RNA.
 7. The methodaccording to claim 1, wherein the polynucleotide is inserted in the formof DNA into a vector in an expressible manner.
 8. The method accordingto claim 1, wherein the polynucleotide is encapsulated into a carrierselected from the group consisting of non-cationic polymer carriers,liposome carriers, dendritic carriers, nano-material carriers,microparticle carriers, biostructural carriers, micelle carriers,polymer microparticles, and magnetic microparticles; or thepolynucleotide is bound to the carrier.